Figure 6

p44/42 MAPK and p70 S6 kinase in cells incubated in the presence of insulin (2.5 μg/ml), PD98059 (10 μM) and/or rapamycin (50 nM). Cells were harvested and immunoblotted for phosphorylated p44/42 MAPK and p70 S6 kinase, then the immunoblots were stripped and reprobed for total MAPK and p70 S6 kinase. The data are representative of two experiments. Protein molecular weight standards (kDa) are shown on the right. (A) When the cells were pretreated with PD98059, the phosphorylation of p44/42 MAPK was greatly decreased in both control and insulin-treated cells after 16 hours. (B) Rapamycin did not affect the phosphorylation of p44/42 MAPK after 16 hours. (C) The phosphorylation of Thr389 in p70 S6 kinase was increased in the presence of insulin after 15 min of incubation and remained increased after 16 hours. Rapamycin abolished the phosphorylation of Thr389 in both control and insulin-treated cells at the 15 min and 16-hour incubation times. (D) Insulin increased the phosphorylation of Thr389 p70 S6 kinase after 15 min and 16-hour incubations. PD98059 did not affect the insulin-induced up-regulation at 15 min. (E) Rapamycin decreased the phosphorylation of Thr421/Ser424 in p70 S6 kinase after 15 min and 16 hours in control and insulin-treated cells. (F) PD98059 added alone caused a slight decrease in phosphorylation of Thr421/Ser424 p70 S6 kinase in control cells treated for 15 min and 16 hours but did not affect the phosphorylation in insulin-treated cells. MAPK = mitogen-activated protein kinase